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Hyperphosphatemia is the primary risk factor for vascular calcification, which is closely associated with cardiovascular morbidity and mortality. Recent evidence showed that oxidative stress by high inorganic phosphate (Pi) mediates calcific changes in vascular smooth muscle cells (VSMCs). However, intracellular signaling responsible for Pi-induced oxidative stress remains unclear. Here, we investigated molecular mechanisms of Pi-induced oxidative stress related with intracellular Ca2+ ([Ca2+]i) disturbance, which is critical for calcification of VSMCs. VSMCs isolated from rat thoracic aorta or A7r5 cells were incubated with high Pi-containing medium. Extracellular signal-regulated kinase (ERK) and mammalian target of rapamycin were activated by high Pi that was required for vascular calcification. High Pi upregulated expressions of type III sodium-phosphate cotransporters PiT-1 and -2 and stimulated their trafficking to the plasma membrane. Interestingly, high Pi increased [Ca2+]i exclusively dependent on extracellular Na+ and Ca2+ as well as PiT-1/2 abundance. Furthermore, high-Pi induced plasma membrane depolarization mediated by PiT-1/2. Pretreatment with verapamil, as a voltage-gated Ca2+ channel (VGCC) blocker, inhibited Pi-induced [Ca2+]i elevation, oxidative stress, ERK activation, and osteogenic differentiation. These protective effects were reiterated by extracellular Ca2+-free condition, intracellular Ca2+ chelation, or suppression of oxidative stress. Mitochondrial superoxide scavenger also effectively abrogated ERK activation and osteogenic differentiation of VSMCs by high Pi. Taking all these together, we suggest that high Pi activates depolarization-triggered Ca2+ influx via VGCC, and subsequent [Ca2+]i increase elicits oxidative stress and osteogenic differentiation. PiT-1/2 mediates Pi-induced [Ca2+]i overload and oxidative stress but in turn, PiT-1/2 is upregulated by consequences of these alterations. [ABSTRACT FROM AUTHOR]

Background/Aims: Fibroblast growth factor (FGF) 21 is associated with hepatic inflammation and fibrosis. However, little is known regarding the effects of inflammation and fibrosis on the β-Klotho and FGF21 pathway in the liver. Methods: Enrolled patients had biopsy-confirmed viral or alcoholic hepatitis. FGF19, FGF21 and β-Klotho levels were evaluated using enzyme-linked immunosorbent assay, realtime polymerase chain reaction, and Western blotting. Furthermore, we explored the underlying mechanisms for this process by evaluating nuclear factor-κB (NF-κB) and c-Jun Nterminal kinase (JNK) pathway involvement in Huh-7 cells. Results: We observed that the FGF19 and FGF21 serum and mRNA levels in the biopsied liver tissue gradually increased and were correlated with fibrosis stage. Inflammatory markers (interleukin 1β [IL-1β], IL-6, and tumor necrosis factor-α) were positively correlated, while β-Klotho expression was negatively correlated with the degree of fibrosis. In Huh-7 cells, IL-1β increased FGF21 levels and decreased β-Klotho levels. NF-κB and JNK inhibitors abolished the effect of IL-1β on both FGF21 and β-Klotho expression. FGF21 protected IL- 1β-induced growth retardation in Huh-7 cells. Conclusions: These results indicate that the inflammatory response during fibrogenesis increases FGF21 levels and suppresses β-Klotho via the NF-κB and JNK pathway. In addition, FGF21 likely protects hepatocytes from hepatic inflammation and fibrosis. [ABSTRACT FROM AUTHOR]

Klotho functions as a tumor suppressor predominantly expressed in renal tubular cells, the origin of clear cell renal cell carcinoma (ccRCC). Altered expression and/or activity of growth factor receptor have been implicated in ccRCC development. Although Klotho suppresses a tumor progression through growth factor receptor signaling including insulin-like growth factor-1 receptor (IGF-1R), the role of Klotho acting on IGF-1R in ccRCC and its clinical relevance remains obscure. Here, we show that Klotho is favorable prognostic factor for ccRCC and exerts tumor suppressive role for ccRCC through inhibiting IGF-1R signaling. Our data shows the following key findings. First, in tumor tissues, the level of Klotho and IGF-1R expression are low or high, respectively, compared to that of adjacent non-neoplastic parenchyma. Second, the Klotho expression is clearly low in higher grade of ccRCC and is closely associated with clinical outcomes in tumor progression. Third, Klotho suppresses IGF-1-stimulated cell proliferation and migration by inhibiting PI3K/Akt pathway. These results provide compelling evidence supporting that Klotho acting on IGF-1R signaling functions as tumor suppressor in ccRCC and suggest that Klotho is a potential carcinostatis substance for ccRCC. [ABSTRACT FROM AUTHOR]

Inorganic phosphate (Pi) plays an important role in cell signaling and energy metabolism. In insulin releasing cells, Pi transport into mitochondria is essential for the generation of ATP, a signaling factor in metabolism-secretion coupling. Elevated Pi concentrations, however, can have toxic effects in various cell types. The underlying molecular mechanisms are poorly understood. Here, we have investigated the effect of Pi on secretory function and apoptosis in INS-1E clonal β-cells and rat pancreatic islets. Elevated extracellular Pi (1~5 mM) increased the mitochondrial membrane potential (ΔΨm), superoxide generation, caspase activation and cell death. Depolarization of the ΔΨm abolished Pi-induced superoxide generation. Butylmalonate, a nonselective blocker of mitochondrial phosphate transporters prevented ΔΨm hyperpolarization, superoxide generation and cytotoxicity caused by Pi. High Pi also promoted the opening of the mitochondrial permeability transition (PT) pore leading to apoptosis, which was also prevented by butylmalonate. The mitochondrial antioxidants, mitoTEMPO or MnTBAP prevented Pi-triggered PT pore opening and cytotoxicity. Elevated extracellular Pi diminished ATP synthesis, cytosolic Ca2+ oscillations, insulin content and secretion in INS-1E cells as well as dispersed islet cells. These parameters were restored following preincubation with mitochondrial antioxidants. This treatment also prevented high Pi-induced phosphorylation of ER stress proteins. We propose that elevated extracellular Pi causes mitochondrial oxidative stress linked to mitochondrial hyperpolarization. Such stress results in reduced insulin content, defective insulin secretion and cytotoxicity. Our data explain the decreased insulin content and secretion observed under hyperphosphatemic states. [ABSTRACT FROM AUTHOR]

Injury to podocytes leads to the onset of chronic renal diseases characterized by proteinuria. Elevated transforming growth factor (TGF)-β in kidney tissue is associated with podocyte damage that ultimately results in apoptosis and detachment. We investigated the proapoptotic mechanism of TGF-β in immortalized mouse podocytes. Exogenous TGF-β1-induced podocyte apoptosis through caspase-3 activation, which was related to elevated ROS levels generated by selective upregulation of NADPH oxidase 4 (Nox4). In mouse podocytes, Nox4 was predominantly localized to mitochondria, and Nox4 upregulation by TGF-β1 markedly depolarized mitochondrial membrane potential. TGF-β1-induced ROS production and caspase activation were mitigated by an antioxidant, the Nox inhibitor diphenyleneiodonium, or small interfering RNA for Nox4. A TGF-β receptor I blocker, SB-431542, completely reversed the changes triggered by TGF-β1. Knockdown of either Smad2 or Smad3 prevented the increase of Nox4 expression, ROS generation, loss of mitochondrial membrane potential, and caspase-3 activation by TGF-β1. These results suggest that TGF-β1-induced mitochondrial Nox4 upregulation via the TGF-β receptor-Smad2/3 pathway is responsible for ROS production, mitochondrial dysfunction, and apoptosis, which may at least in part contribute to the development and progression of proteinuric glomerular diseases such as diabetic nephropathy. [ABSTRACT FROM AUTHOR]

Klotho is a mammalian senescence-suppression protein that has homology with glycosidases. The extracellular domain of Klotho is secreted into urine and blood and may function as a humoral factor. Klotho-deficient mice have accelerated aging and imbalance of ion homeostasis. Klotho treatment increases cell-surface abundance of the renal epithelial Ca2+ channel TRPVS by modifying its N-linked glycans. However, the precise sugar substrate and mechanism for regulation by Klotho is not known. Here, we report that the extracellular domain of Klotho activates plasma-membrane resident TRPV5 through removing terminal sialic acids from their glycan chains. Removal of sialic acids exposes underlying disaccharide galactose-N-acetylglucosamine, a ligand for a ubiquitous galactoside-binding lectin galectin-1. Binding to galectin-1 lattice at the extracellular surface leads to accumulation of functional TRPV5 on the plasma membrane. Knockdown of β-galactoside α2,6-sialyltransferase (ST6GaI-1) by RNA interference, but not other sialyltransferases, in a human cell line prevents the regulation by Klotho. Moreover, the regulation by Klotho is absent in a hamster cell line that lacks endogenous ST6GaI-1, but is restored by forced expression of recombinant ST6GaI-1. Thus, Klotho participates in specific removal of α2,6-linked sialic acids and regulates cell surface retention of TRPV5 through this activity. This action of Klotho represents a novel mechanism for regulation of the activity of cell-surface glycoproteins and likely contributes to maintenance of calcium balance by Klotho. [ABSTRACT FROM AUTHOR]

Transient receptor potential vanilloid 5 (TRPV5) constitutes the apical entry pathway for transepithelial Ca2+ reabsorption in kidney. Many hormones alter renal Ca2+ reabsorption at least partly by regulating TRPV5. The mechanism for acute regulation of TRPV5 by phospholipase C-coupled hormones is largely unknown. Here, we found that protein kinase C (PKC) activator 1-oleoyl-acetyl-sn-glycerol (OAG) increased TRPV5 current density and surface abundance in cultured cells. The OAG- mediated increase of TRPV5 was prevented by preincubation with specific PKC inhibitors. Coexpression with a dominant-negative dynamin increased the basal TRPV5 current density and prevented the increase by OAG. Knockdown of caveolin-l by small interference RNA (siRNA) prevented the increase of TRPV5 by OAG. In contrast, knockdown of clathrin heavy chain had no effects. OAG had no effect on TRPV5 expressed in caveolin-I null cells derived from caveolin-1 knockout mice. Forced expression of recombinant caveolin- I restored the regulation of TRPV5 by OAG in caveolin- I knockout cells. Mutations of serine-299 and/or serine-654 of TRPV5 (consensus residues for phosphorylation by PKC) abolished the regulation by OAG. Parathyroid hormone (PTH) increased TRPV5 current density in cells coexpressing TRPV5 and type I PT!-! receptor. The increase caused by PTH was prevented by PKC inhibitor, mutation of serine-299/serine-654, or by knockdown of caveolin- I. Thus, TRPV5 undergoes constitutive caveolae-mediated endocytosis. Activation of PKC increases cell surface abundance of TRPV5 by inhibiting the endocytosis. This mechanism of regulation by PKC may contribute to the acute stimulation of TRPV5 and renal Ca2+ reabsorption by PTH. [ABSTRACT FROM AUTHOR]

The transient receptor potential channel TRPV5 contributes to the apical entry pathway for transcellular calcium reabsorption in the kidney. Acid load causes hypercalciuria in animals and humans. We have previously reported that intracellular protons directly inhibit TRPV5. Here, we examined the effects of intracellular pH on single-channel activity of TRPV5. We found that TRPV5 channels exhibit full and subconductance open states in excised inside–out patches of Chinese hamster ovary cells. The slope conductance values (Na+ as a charge carrier, between −25 and −75 m V) for full and subconductance opening at intracellular pH 7.4 were 59 ± 6 and 29 ± 3 pS, respectively. Intracellular acidification caused a small decrease in single-channel conductance. Importantly, intracellular acidification decreased open probability for the full and subconductance states and increased probability for closing. To investigate how intracellular protons decrease open probability of the channel, we proposed a simple three-state model for open–subconductance–closed state transition and examined the effects of acidification on the respective forward and reverse rate constants. We found that intracellular acidification decreases opening of TRPV5 predominantly by promoting a transition from the subconductance to the closed state. Thus, intracellular acidification directly inhibits TRPV5 by causing a conformational change(s) leading to a decrease of open probability of TRPV5 as well as of the single-channel conductance. [ABSTRACT FROM AUTHOR]